Biochemical tests /Api (20) E system Dr. Suhad Faisal Hatem

The API 20E fast identification system combines some conventional tests and allows the identification of a limited number of Gram-negative Enterobacteriaceae. The identification of most Gram-negative bacilli is far more complex and often requires panels of 20 tests in small reaction tubes, which include the substrates in addition to oxidase test which consider no.21. An identification is only possible with microbiological culture.

API test plastic strips consists of wells or mini-test chambers containing dehydrated substrates to detect enzymatic activity, usually related to fermentation of carbohydrate or catabolism of proteins or amino acids by the inoculated organisms. A bacterial suspension is used to rehydrate each of the wells and the strips are incubated. During incubation (18-24h. Incubation at 35-37C), metabolism produces color changes that are either spontaneous or revealed by the addition of reagents. For example, when carbohydrates are fermented, the pH within the well decreases and that change is indicated by a change in the color of the pH indicator. All positive and negative test results are compiled to obtain a profile number, which is then compared with profile numbers in a commercial codebook to determine the identification of the bacterial species. 3

Procedure 1-Prepare a saline suspension from the center of the colony on an agar plate to the tube of normal saline , then vortex it . 2-Dispense about 5 ml of sterile water into tray. 3-Fill the micropipette and inoculate all the tubes on the strip. 4-Slightly under fill ADH , LDC , ODC ,H2S, URE to provide anaerobic conditions for these compartment, dispense mineral oil to the cupules. 5-Fill both the tube and cupules of the test CIT,VP, GGL with the bacterial suspension, these wells require O2 .Also fill all wells bacterial suspension aerobically. 6-Incubation the strip at 37οC for 18-24.After incubation some tests need the additional reagents as follow:

A-VP test: add one drop of VP1 &VP2 reagent and wait for 10 min A-VP test: add one drop of VP1 &VP2 reagent and wait for 10 min. before record the result. B-TDA test: add one drop of TDA reagent , the +ve result (brown-red) color will occur immediately. C-IND test: add one drop of IND reagent and wait for 2 min. before record the result. 7-All reactions will recorded as number for +ve and –ve results on the report sheet. 7 digital profile number will be determined , so the unknown organism can be looked up in the API 20E analytical profile index.

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